From field to genome: non-lethal, on-site nanopore-only assembly of the endangered Grenada Frog (Pristimantis euphronides).

Abstract

Background: The genus Pristimantis is among the most species-rich vertebrate genera, with more than 600 described species, yet no whole-genome assembly was available for any member of the genus or for the families Strabomantidae or Craugastoridae prior to this work. The Grenada Frog (Pristimantis euphronides) is a critically endangered direct-developing anuran endemic to montane rainforest on Grenada in the Lesser Antilles. Reference genomes are foundational tools for the conservation of endangered species, but their production typically depends on destructive sampling, large institutional infrastructure, or the shipment of biomaterials abroad.

Results: We present a 1.75 Gb scaffold-level reference genome of P. euphronides assembled from a single wild female sampled non-lethally by toe clip, blood swab, and buccal swab. All wet-laboratory steps, including Oxford Nanopore Technologies (ONT) MinION sequencing, were performed in-country on portable equipment, and downstream analyses ran on laptop-class hardware and a remote server accessed from Grenada. The Flye-Medaka assembly, scaffolded with RagTag against Eleutherodactylus coqui, comprised 13 pseudochromosomes with a scaffold N50 of 117.8 Mb, achieved 91.4% tetrapod BUSCO completeness, and supported 28,071 annotated protein-coding genes. Cross-species synteny analysis against three additional Hyloidea genomes computationally predicted an underlying karyotype of n = 16, consistent with prior cytogenetic counts, indicating that the reference-guided pseudochromosomes do not correspond to the true haploid chromosome number. Coverage analysis identified candidate Z-linked regions on two scaffolds and a set of unplaced contigs with sequence features consistent with the heterochromatic W chromosome described from C-banding, providing a first-pass framework for sex chromosome analyses in Pristimantis.

Conclusions: This whole-genome assembly provides a reference for phylogenomic and conservation genomic studies in a hyperdiverse direct-developing frog clade in which previous comparative work had relied on few marker genes. In the absence of chromatin conformation capture methods such as Hi-C, which were not compatible with non-lethal field sampling, reference-guided scaffolding against the closest available chromosome-scale Hyloidea genome, approximately 52 million years divergent, provided a workable alternative. The non-lethal, in-country, ONT-only workflow demonstrates that scaffold-level reference genomes for repeat-rich vertebrates of conservation concern can be produced without destructive sampling or outsourcing to external sequencing or bioinformatics services.